The overall objective of this Project is to examine and optimize combination immunization strategies that effectively induce both antibody and CD8 +T cell mediated immune responses against primate lentiviruses. Existing evidence indicates that multiple mechanisms, including neutralizing antibodies and CD8+ T-mediated immune responses, contribute to protection and control of virus infection, Since different immunization modalities activate different responses, we hypothesize that combination of immunization modalities (" prime and boost") offers the best likehhood of inducing the balanced immune responses required for vaccine protection. The synergistic effect of priming with recombinant vaccinia virus followed by boosting with protein vaccines was first demonstrated in Project Leader's laboratory in mice. Augmentation of HIV specific antibody response by a similar strategy was subsequently observed in humans and the protective efficacy of this approach demonstrated, first in an SIV and later in an SHIV model. Since then a number of "prime and boost" strategies have been developed, including DNA/MVA, DNA/protein, and other combinations. Induction of strong CD8 T-cell mediated responses and partial or complete protection against other primate lentiviruses have been reported. However, the basis of the synergistic effect observed in prime and boost strategies and approaches to optimize both antibody and CD8+ T-cell mediated responses have not been fully explored. In this Project, we aim to address these issues in a systematic study of the three most commonly used modalities of immunization: poxvirus vectors, DNA vaccines and subunit proteins. Novel formulation and delivery approach for each of these modalities will be included. Immune responses for each modality individually and m combinations will be compared and analyzed first in mice. Approaches that elicit the best responses as measured by one or more parameters will be selected for evaluation in macaques, both for immunogenicity and for protection against SHIV infection. Finally, the best envelope antigen designs from Projects 1-3 will be combined with the most promising immunization approach(es) identified in this Project to evaluate their protective efficacy against homologous and heterologous SHIV challenges. This Project will also participate in the evaluation of T-cell mediated immune responses in vaccine and challenge studies described in Projects 2 and 3, thus providing further insights into effective vaccine designs and correlates of protective immunity. The Specific Aims of this Project are: 1. Analysis and optimization of single and mixed modality immunization approaches in mice 2. Analysis and optimization of mixed modality immunization approaches in macaques 3. Evaluation of protective efficacy of mixed modality immunization approaches in macaques 4. Evaluation of immune responses and protective efficacy of multigenlc vaccines with optimized envelope designs and vaccination approaches 5. Analysis of cellular immune responses in vaccine and challenge studies proposed in Projects 2 and 3. This Project will interact closely with all other Projects and Cores in this Program.